Hepatitis C virus (HCV) infection is a globally devastating disease. Beyond the limited efficiency combination treatment of 1-interferon (native or pegylated) and the nucleoside Ribavirin, which was introduced in 1990, there has not been another drug registered in the following 20 years. HCV NS5B polymerase is a validated target essential to virus replication. Currently, after the failure of several nucleosides and nucleoside prodrugs in HCV clinical trials, five nucleoside monophosphate prodrugs that target the NS5B polymerase are in HCV clinical trials. In this application, we will demonstrate that delivery of stabilized species of activated triphosphates of nucleosides (nucleoside triphosphate mimics, N3PMs) as prodrugs (N3PM-PDs) will be superior as inhibitors of HCV replication to those corresponding nucleosides, nucleoside prodrugs and nucleoside monophosphate prodrugs that have entered clinical trials. To accomplish this, we introduce several innovations in this application. 1) a structure- activity-relationship (SAR) study will identify the most active (against HCV NS5B polymerases) and selective (with lack of activity towards human polymerases) ribofuranosyl-triphosphate (2,3-CF2-P-1-P-modified-3'-obligate modified ribofuranosyl) hetero-bases;2) obligate chain-termination ribofuranosyl moieties will be explored rather than the popular 2'-C-Me ribofuranosyl sugar employed by current clinical nucleosides;3) a non-Watson/Crick, non-ionic, lipophilic, alternative hetero-base will be initially examined;4) the optimized ribofuranosyl-triphosphate (2,3-CF2-P-1-P-modified-3'-obligate modified ribofuranosyl) will be generic for inhibition of HCV NS5B. Thus, various hetero-bases (heterocycles) conjugated to the SAR optimized ribofuranosyl-triphosphate can be examined;and 5) FDA-approved pivaloyloxymethyl (POM) prodrugs of these N3PMs will allow passive diffusion uptake and intracellular delivery of high concentrations of active species of N3PMs. Clearly, other literature ribofuranosyl nucleobases exhibiting interesting anti-HCV activity could be examined by our discovery process. For AIM 1 of this proposal, we consider the clinically validated POM moiety as a "generic" prodrug for N3PMs. Its use will allow structure-activity relationship studies in HCV replicons to move as rapidly as possible to determine the optimal 1-P and ribofuranosyl modifications of various hetero-bases. Optimizing the most effective prodrug will be a key AIM in a subsequent Phase 2 SBIR. In particular, consideration of liver-targeting prodrug moieties that may provide higher concentration of N3PMs in liver tissue will be explored. This approach alows us to move very rapidly, efficiently and economically in an effort to discover superior HCV inhibitors suitable for preclinical development, consistent with a recognized need for facilitated translational research. PUBLIC HEALTH RELEVANCE: Hepatitis C virus is global devastating disease, with minimal, effective therapeutics. We will demonstrate that the delivery of stabilized active species of nucleosides (nucleoside triphosphate mimics, N3PMs) as prodrugs (N3PM-PDs) will be superior as inhibitors of HCV replication than corresponding nucleosides, nucleoside prodrugs and nucleoside monophosphate prodrugs that have entered clinical trials. This approach allows us to move very rapidly, efficiently and economically in an effort to discover superior HCV inhibitors suitable for preclinical development, consistent with a recognized need for facilitated translational research.